Solid supports for nucleic acid hybridization assays

ABSTRACT

Compositions and methods for covalently immobilizing an oligonucleotide onto a polymer-coated solid support or similar structure are provided. Specifically, the polymer-coated support, such as a bead, possesses a large number of activatable moieties, preferably primary and secondary amines. An oligonucleotide is activated with a monofunctional or multifunctional reagent, preferably the homotrifunctional reagent cyanuric chloride. The resultant covalently immobilized oligonucleotides on the support serve as nucleic acid probes, and hybridization assays can be conducted wherein specific target nucleic acids are detected in complex biological samples. The beads or similar structures can be employed free in solution, such as in a microtiter well format; in a flow-through format, such as in a column; or in a dipstick. Additionally, dichlorotriazine oligonucleotides and processes for activating oligonucleotides by treatment with cyanuric chloride and derivatives are included in the present invention.

RELATED CASES

This application is a continuation of application Ser. No. 08/341,465,filed Nov. 16, 1994 now abandoned which is a continuation of Ser. No.07/907,931 filed Jun. 25, 1992 now abandoned which is acontinuation-in-part of Ser. No. 07/522,442 filed May 11, 1990 nowabandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of nucleic acidhybridization assays for detecting specific polynucleotide sequencesand, more particularly, to compositions and methods for covalentlyattaching activated oligonucleotides to polymer-coated beads serving assolid supports.

Nucleic acid hybridization is a known method for identifying specificsequences of nucleic acids. Hybridization is based upon base pairingbetween complementary nucleic acid strands. When single stranded nucleicacids are incubated in appropriate buffer solutions, complementary basesequences pair to form double stranded stable molecules. The presence orabsence of such pairing may be detected by several different methodsdescribed in the art.

Many known hybridization assays involve multiple steps, for example thehybridization technique described by Dunn, et al., Cell 12:23-36 (1977)(incorporated herein by reference), wherein a sandwich-type assayconsists of a first hybridization between a "target" nucleic acid and a"capture" nucleic acid probe that has been immobilized on a solidsupport and a second hybridization between a "signal" nucleic acidprobe, typically labeled with a radioactive isotope, and a differentregion of the immobilized target nucleic acid. The hybridization of thesignal probe may then be detected by, for example, autoradiography.

Ranki, et al., U.S. Pat. No. 4,486,539 and U.S. Pat. No. 4,563,419 (bothpatents incorporated herein by reference), describe sandwich-type assaysthat first require steps to render nucleic acids single stranded beforethe single stranded nucleic acids are allowed to hybridize with anucleic acid affixed to a solid carrier and with a nucleic acid labeledwith a radioisotope.

Carrico, et al., U.S. Pat. No. 4,806,546, and Carrico, et al., EuropeanPatent Application 86112899.9 (both incorporated herein by reference),have described treatment of a nylon support with an alkylating agent tointroduce amidine groups onto the surface of the nylon. The derivednylon surface possesses the capacity to noncovalently bind singlestranded nucleic acids. The noncovalently bound nucleic acids are thenused as probes to detect specific target nucleic acids in solution.

Hunger, et. al., Analytical Biochemistry 165:45-55 (1987); Hunger, etal., Analytical Biochemistry 156:286-299 (1986); Hunger, et al.,European Patent Application 84109485.7 (all incorporated herein byreference), describe the use of cyanuric chloride-activated cellulosepaper having immobilized restricted genomic DNA in Southern blottechniques for the detection of subpicogram quantities of complementaryDNA. Biagioni, et. al., Analytical Biochemistry 89:616-619 (1978)(incorporated herein by reference), describe a method for thepreparation of DNA-cellulose using cyanuric chloride wherein theDNA-cellulose is employed in affinity chromatography procedures.

Herzberg, et al., European Patent Application 0171150 (incorporatedherein by reference), describe the use of oligonucleotides immobilizedonto solid supports in dipstick assays.

Litman, et al., U.S. Pat. No. 4,391,904 (incorporated herein byreference, describes test strip kits wherein a member of animmunological pair is bonded to a solid surface. Also, Miller, et. al,Clin. Chem. 30:1467-1472 (1984), and Brown, et. al, Clin. Chem.31:1500-1505 (1985) (both are incorporated herein by reference),describe an analytical test chamber containing cellulose threads coupledto an antibody as a solid matrix that permits multiple test results froma single sample.

SUMMARY OF THE INVENTION

The present invention comprises novel compositions and processes havingutility in nucleic acid hybridization assays and other fields. Accordingto one aspect of this invention, compositions comprising an activatedoligonucleotide, which can be covalently attached to a polymer-coatedsupport such as a bead are described. Preferably, cyanuric chloride, ahomotrifunctional reagent, activates the oligonucleotide, or derivativesof cyanuric chloride. Processes for covalently immobilizing activatedoligonucleotides on solid supports, such as nylon, latex beadscontaining amino, carboxyl, sulfonic or hydroxyl groups, polystyrenecoated magnetic beads containing amino or carboxylate groups, glass,teflon, or plastic are also included herein.

The covalently immobilized oligonucleotides on polymer-coated beads orsimilar structures can serve as nucleic acid probes, and hybridizationassays can be conducted wherein specific target nucleic acids aredetected in complex biological samples. The beads can be employed freein solution, such as in a microtiter well format; in a flow-throughformat, such as in a column; or in a dipstick. Additionally, immobilizedoligonucleotides on a polymer-coated bead can be of the same ordifferent nucleic acid sequences.

The bead solid supports of the present invention possess the followingadvantages over known membrane or bead supports, in which capturenucleic acid sequences are noncovalently attached. First, the capturerate of target nucleic acid sequences is improved 5 to 25-fold, and 100%of the capture nucleic acid sequence is available for hybridization witha complementary sequence; second, the quantity of immobilized capturenucleic acid can be increased approximately 20-fold on an apparentsurface area basis; third, greater ease of manufacturing exists; fourth,the bead possesses covalently immobilized, capture nucleic acidsequences (oligonucleotides) and can withstand denaturation temperaturesin excess of 90° C. for 10 or more minutes; and, finally, a multisitedipstick can be constructed, leading to miniaturization of a detectiondevice. All of these advantages contribute to greater sensitivitieswhen, for example, a sandwich assay format is used.

Dipsticks are also included in the present invention. These dipstickscomprise a nonporous solid support and a means for attaching the beadsor similar structures discussed above. Additionally, multiple beadspossessing oligonucleotides with different sequences or specificitiescan be closely aligned on a multisite dipstick, giving rise to anindicator card that can detect a multiplicity of pathogens in a singlebiological example, for example, to identify bacterial and viral agents.

The use of beads or similar structures in the dipstick format achieves asignificant decrease in nonspecific background levels of signal systemsbecause of the simple pressure fit by which the bead or similarstructure is placed in the dipstick, as compared with membrane supportsand the like, which necessarily must be sandwiched between two supportsor glued or attached in place.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and to the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a configuration for a dipstick of the presentinvention;

FIG. 2 is a multisite indicator card that can be used for periodontitisdiagnosis; and

FIGS. 3A, 3B, 3C and 3D high performance liquid chromatography (HPLC)profiles showing that cyanuric chloride reacts selectively with the5'-tethered amine of an oligonucleotide, and not with sugars or basesthereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes compositions useful in nucleic acidhybridization assays. These compositions comprise a dichlorotriazineoligonucleotide and other activated oligonucleotides covalently attachedto a polymer-coated bead or similar structure.

Additionally, processes for covalently immobilizing an oligonucleotideon a solid support, preferably a bead, are included. In general, theseprocesses comprise the steps of treating a solid support with analkylating agent; reacting the treated solid support with anamine-containing polymer, whereby the polymer covalently coats the solidsupport; activating an oligonucleotide with a monofunctional ormultifunctional reagent, preferably the homotrifunctional reagent,cyanuric chloride (i.e., 2,4,6-trichlorotriazine); conjugating theactivated oligonucleotide and the polymer-coated solid support. Theunreacted amines are then blocked by acylation to impart the propersurface charge to the solid support surface.

The term "solid support" refers to any surface that is transferable fromsolution to solution or forms a structure for conductingoligonucleotide-based assays, and includes beads, membranes, microtiterwells, strings, plastic strips, or any surface onto which nucleic acidprobes may be immobilized.

As used herein, "bead" encompasses any type of solid or hollow sphere,ball, bearing, cylinder, or other similar configuration composed ofplastic, ceramic, metal, or polymeric material onto which a nucleic acidcan be covalently immobilized. As such, the term also includes string orstrings. Preferably, a bead that is spherical in shape is employed inthe present compositions, and a preferred diameter range for such beadsis from about 0.01 inch to about 0.5 inch, more preferably from about0.06 inch to about 0.09 inch (corresponding to commercially available3/32 inch nylon beads), and most preferably about 0.09 inch(corresponding to commercially available 3/32 inch nylon beads).Additionally, it is preferred that the beads are unpolished or, ifpolished, roughened before treating with an alkylating agent.

In the present invention the solid support will typically be comprisedof a polymeric material containing derivatizable functional groups[e.g., poly(p-aminostyrene)] or polymeric soild supports that can beactivated (e.g., nylon beads, cyanuric chloride activated cellulosecommercially available). Examples of preferred solid supportcompositions include nylon, polystyrene, glass, polypropylenes,polystyrene/glycidyl methacrylate latex beads, latex beads containingamino, carboxyl, sulfonic and/or hydroxyl groups, polystyrene coatedmagnetic beads containing amino and/or carboxylate groups, glass,teflon, plastic and the like.

Procedures for preparing the different solid phases will be differentdepending on the functional groups available on the solid support andthe polymer coat. The chemistries to covalently couple compounds withthe same or different functional grouops are well known in the art. See,e.g., Ji, Method. Enzymol. 91:580-609 (1983), which is incorporatedherein by reference. For example, a nylon bead or beads, or anycomposition or structure of nylon, is first derived (prepared) bytreating the bead with an alkylating agent. Alkylating agents used inthis manner react with amides present in the nylon to form a reactiveimidate ester. Preferred alkylating agents are described below. Theactivated nylon surface is reacted with a polymer containing an aminegroup, and the coated bead is then reacted with cyanuric chloride orderivatives as appropriate. For solid supports that contain reactivefunctional groups, the appropriate chemistries, well known to theskilled artisan, are selected to coat the bead. To immobilize theaminohexyl tailed oligonucleotides to the polymer coated solid supportrequires the appropriate chemistries, depending on the functionalgroups. Most solid support surfaces described above can be modified tointroduce polar groups on the surface by a process referred to as theCoronal Discharge Process. Using this process, a support, e.g., plastic,is subjected to sparks of electricity in the presence of ozone tointroduce carboxyl groups.

Preferred alkylating agents include, but are not limited to, dialkylsulfates, alkyl triflates, alkyldiphenyl sulfonium salts, alkylperchlorates, and, preferably, trialkyloxonium salts. The latterincludes the lower alkyl salts, trimethyloxonium and triethyloxoniumsalts. The salt counterion can be selected from the group consisting ofhexachloroantimonate, hexafluorophosphate, and tetrafluoroborate, withthe last named counterion being preferred; however, other saltcounterions can also be used and will be apparent to one skilled in therelevant art.

The selection of a solvent for the alkylating agent is important for thepresent invention. A solvent should be employed that does not dissolveor render tacky nylon during the alkylation. Non-nucleophilic organicsolvents, such as dichloromethane, dimethylsulfoxide, tetrahydofuran,N-methyl-pyrrolidone, and others are appropriate solvents. Inparticular, N-methyl-pyrrolidone is preferred.

The resulting imidate esters on the bead surface are then reacted undersuitable conditions with an amine-containing polymer, whereby amidineresidues are formed. Any primary or secondary amine-containing polymercan be employed to form amidine residues, thus covalently immobilizingthe polymer onto the surface of the bead. Poly(ethyleneimine) (PEI),polyallylamine, and polyvinylamine are preferred examples. PEI isavailable in a variety of molecular weights, ranging from about 600 toabout 100,000 Daltons, although preferably a polymer of about 30,000 MWis used to coat a solid phase. A PEI polymer of 10,000 MW contains about58 primary, 116 secondary and 58 tertiary amine groups, and is abush-like structure. Examples of polymers which introduce multiplefunctional groups which increases the number of oligonucleotides thatcan be immobilized on a polymer coated surface include cellulosepropionate, poly(acrylamide-acrylic acid 10% carboxyl),poly(acryhydrazide), poly(p-aminostyrene), poly(ethylene-methacrylicacid), poly-L-lysine (MW 40-60,000), poly(vinylsulfonic acid) and thelike. Standard chemistries are used to immobilize polymers (amine,carboxyl, sulfonyl and hydroxyl containing) to amine, carboxyl, sulfonylor hydroxyl containing solid supports. For example, a preferred solventused to dissolve a polymer during the conjugation of the polymer to anactivated nylon bead is N-methyl-pyrrolidone.

Nylon can also be partially hydrolyzed to yield reactive amine orcarboxyl groups that can be reactive with amine- or carboxyl-containingpolymers. Similarly, any carboxyl moieties coating the surface of asolid support can be coated with amine-containing polymers using similarchemistries described above.

In addition, any other polymer capable of being derived with any primaryor secondary aliphatic or aromatic amine is suitable for the presentinvention. The units of such a polymer are joined together by directpolymerization process bonding or by coupling agent linking. Directpolymerization produces interbonding of available chemical groups orbackbone moieties in adjacent units. For example, oxidative enzymes canbe used to polymerize monomer units by oxidative cross-linking.Alternatively, a coupling agent, derived from a bifunctional ormultifunctional organic cross-linking agent, can bond with theappropriate chemical group or backbone moiety of the units. In thiscontext the term "coupling agent" denotes the linkage group afterbonding and the term "cross-linking reagent" denotes the linkagecompound before bonding.

The cross-linking reagent has generic formula: ##STR1## wherein reactivegroups A, B, and E are independently selected from the group consistingof hydrogen, a carboxylic acid group, an acid halide, an activatedester, a mixed anhydride, an iminoester, a primary amine, an aldehydegroup, an ∝-halo methylcarbonyl group, a hydrazine group, an acylhydrazide group, an azide group, and an N-maleimide group, wherein atleast two of A, B, and E are other than hydrogen. R¹ is an aliphaticgroup of at least two carbons or an aromatic or heterocycle group of atleast six carbons.

Multifunctional cross-linking reagents, with more than three reactivegroups that are similar to A, B, and E are also within the scope of thepresent invention. These additional reactive groups will beindependently selected from the foregoing definitions of A, B, and E.

Choice of the reactive groups will depend upon the selection of thechemical groups or backbone moieties of the polymer units (monomericunits) that are to be linked. Each type of chemical group or backbonemoiety will react with the appropriate reactive group or groups. Forexample, an amine group will react with a carboxylic acid group, an acidhalide, an activated ester, a mixed anhydride, an acyl imidazolide, anN-(carbonyloxy)imide group, an iminoester, or an aldehyde group. Anoxidized 1,2-diol group (a dialdehyde) will react with a primary amine,a hydrazine group, an azide, or an acyl hydrazide group. A carbonylgroup will react with a primary amine, a hydrazine group, or an acylhydrazide group. A mercaptan group will react with a carboxylic acidgroup, an acid halide, an activated ester, a mixed anhydride, an acylimidazolide, or an N-(carbonyloxy)imide. A carbon-hydrogen bond willreact with an azide (nitrene). The solid supports are thus coated withthe selected polymer, including multifunctional polymers, that containsa large number of activatable primary and secondary amines.

The table below illustrates, but does not limit, the types of reactionsthat may be employed in the present invention to covalently attach,either directly or through a spacer arm, a polymer to a solid support.As can be seen, although amine-containing polymers are preferred, otherpolymers containing thiol or carboxyl groups can be employed as well.

    ______________________________________                                        Bead         Polymer      Coupling                                            functionality:                                                                             functionality:                                                                             reagent:                                            ______________________________________                                        Amine        Amine        Cyanuric chloride                                   Amine        --COO.sup.-  Carbodiimide                                        Amine        --SH         Heterobifunctional                                  --COO.sup.-  Amine        Carbodiimide                                        --OH         Amine        Cyanuric chloride                                   ______________________________________                                    

The polymer-coated solid supports are then conjugated with activatedoligonucleotides or other substances of interest using similar oridentical chemistries to those described above. Additionally, anamine-containing polymer can be covalently linked through the amine to aprotein by activating the protein with a homofunctional orheterofunctional reagent and conjugating the activated protein to thepolymer. The protein may be a receptor, ligand, enzyme, hormone,interleukin, antibody, etc. Conveniently, the polymer is coupled to asolid support, such as a bead, membrane, microwell, centrifuge tube, orstring.

As used herein, oligonucleotides refer to short nucleic acid sequencesthat are approximately 12 to 100 bases in length. Such oligonucleotidescan be used as capture probes in hybridization assays and are preferablychemically synthesized using commercially available methods andequipment. For example, the solid phase phosphoramidite method can beused to produce short probes of between 15 and 50 bases having amolecular weight of less than 16,000 daltons. For the synthesis ofoligonucleotides, see Caruthers, et al., Cold Spring Harbour Symp.Quant. Biol. 47:411-418 (1982); Adams et al., J. Am. Chem. Soc. 105:661(1983) (both are incorporated herein by reference).

When synthesizing an oligonucleotide probe for a specific target nucleicacid, the choice of nucleotide sequence will determine the specificityof the test. For example, by comparing DNA sequences from severalbacterial isolates, one can select a sequence for bacterial detectionthat is either type-specific or genus-specific. Comparisons of DNAregions and sequences can be achieved using commercially availablecomputer programs.

The preferred capture oligonucleotides for use in the present inventionare synthetic oligonucleotides from about 16 to about 100 bases inlength. A spacer (linker) arm, i.e., a chemical moiety that extends orlinks other chemical groups, and preferably is a carbon chain containingfrom about 2 to about 12 carbon atoms, more preferably about 6 carbonatoms, containing a blocked amine group can be coupled during synthesisusing conventional chemistry to the 5'-hydroxyl group of anoligonucleotide. A primary amine is the preferred group for attachmentto monofunctional or multifunctional reagents, and its attachment via ahexyl arm is preferred. The reagents for the attachment of primaryspacer arms terminating in a primary amine are commercially available.Starting materials suitable for use in the present invention aredescribed in PCT 86/01290; Nucl. Acids Res. 15:3131 (1987); Nucl. AcidsRes. 15:2891 (1987); and Nucl. Acids Res. 14:7985 (1986) (allincorporated herein by reference).

Preferably, an oligonucleotide possessing a 5'-terminal structure suchas ##STR2## is employed wherein, as a spacer arm, n is 2-12 inclusive,preferably 6; X is --NH-- or --NHC:O(CH₂)_(m) NH--, preferably --NH--;wherein m is 2-12, inclusive; Y is a 4,6-dichlorotriazine (preferred) orthiol (sulfhydryl) reactive moiety; and A is an oligonucleotide, rangingfrom between about 9-50 bases, preferably between about 15-30 bases,with only the 5'-hydroxyl requiring modification for attachment.

Alternatively, an oligonucleotide can be modified at the 3'-end with aspacer arm containing a blocked amine group. This can be accomplished byconducting DNA synthesis on a solid support containing a conjugatedribonucleotide. After removal from the solid support, a DNAoligonucleotide is obtained that contains a single 3'-terminalribonucleotide. This can be modified with a spacer arm containing anucleophilic amine by, for example, oxidizing the ribonucleotidecis-glycol with periodate; treating oligonucleotide so modified with,for example, butane diamine to form a Schiff base; and treating withsodium borohydride or cyanoborohydride to form a stable reduced Schiffbase derivative in which one of the amines is left free for subsequentconjugation.

The selected oligonucleotides are then activated with a monofunctionalor multifunctional reagent. Activated oligonucleotides refer in generalto oligonucleotides that have been reacted with a chemical compound andrendered chemically active. As used herein, activatable refers to thepotential to become chemically reactive. Multifunctional reagentsinclude, but are not limited to, homotrifunctional, heterotrifunctional,homobifunctional, and heterobifunctional reagents.

Activated oligonucleotides may be linked to polymer-coated solidsupports according to the following chemistries. In general, there aretwo modes by which the oligonucleotide can be covalently attached to thepolymer at this point. An amine-tailed oligonucleotide can be activatedwith a monofunctional or multifunctional reagent, for example cyanuricchloride whereby an alkylamino dichlorotriazine is formed, which is thenreactive toward the amine-containing polymer. Alternatively, the polymeron the surface of the bead can be activated with a reagent, preferablythe homotrifunctional reagent cyanuric chloride, which is then reactivetoward the amine-tailed or amine-derived oligonucleotide.

Although cyanuric chloride, a homotrifunctional reagent is preferred,other reagents can be used. For example,N-succinimidyl-4-(iodoacetamido)-benzoate (SIAB) is a heterobifunctionalreagent, and disuccinimidyl suberate is a homobifunctional reagent. Ifcarboxyl groups are involved, the heterobifunctional reagent,1-ethyl-3-(dimethylaminopropyl)carbodiimide can be used. Other similarmonofunctional and multifunctional (heteromultifunctional andhomomultifunctional) reagents are included for use in processes of thepresent invention.

The chemistries employed in the present invention result in theselective activation of an amino group on an oligonucleotide, withoutmodification of any of the purine and pyrimidine bases of theoligonucleotide, as demonstrated in Example 1 below. The placement ofthe amine-containing polymer on the bead surface and the covalentimmobilization of an activated capture oligonucleotide onto such asurface increases the rate or extent of capture of target nucleic acid 5to 25-fold compared with diamine compounds, for example, hexanediamine.Additionally, the nonspecific binding of biological material on thesurface of the bead is substantially reduced.

The preferred chemistry employs cyanuric chloride (i.e.,2,4,6-trichlorotriazine). The chemistry of the cyanuric chloridereaction is as follows. ##STR3## wherein R is an oligonucleotide andpreferably is of the structure depicted for a 5'-amino hexyloligonucleotide.

Oligonucleotides possessing a 5'- or 3'-tethered (via a hexyl arm)nucleophilic amine moiety (or internal aminoalkyl groups substituted onpyrimidine or purine bases) are reacted with an excess, preferably fromabout 50 to about 200-fold, more preferably 125-fold, of recrystallizedcyanuric chloride at, preferably, 15°-50° C., more preferably at 19°-25°C., in a part organic solvent for a preferred time period from about 30minutes to about 6 hours, more preferably from about 1 to about 2 hours.

A number of other cyanuric chloride derivatives and multi-functionalactivation reagents are available for the activation of amino-tailedoligonucleotides. Representative cyanuric chloride derivatives include2-methoxy-4,6-dichlorotriazine (synthesized according to Schaeffer et al(J. Am. Chem. Soc. 73:2990-2989 (1951)), 2,4,6-tribromotriazine,2,4,6-triiodotriazine, 2-monochloro-4,6-diiodotriazine, and2-amino-4,6-dichlorotriazine.

The rate of reaction of these cyanuric chloride derivative reagents withamino-tailed oligonucleotides may be controlled as necessary, such as byperforming reactions at lower temperatures. For example,2-methoxy-4,6-dichlorotriazine is more reactive than cyanuric chlorideand is prone to hydrolysis by water. A similar compound,2,4-dichlorotriazinyl-cellulose, reacts directly with nucleic acids,presumably through the nucleophilic groups on the purine and pyrimidinebases (Hunger et al., Anal. Biochem. 156:286-299 (1986) and Biagoni etal., Anal. Biochem. 89:616-619 (1978)), which would lead to seriousdecreases in hybridization efficiency with the short syntheticoligonucleotides. It has previously been shown (Hodgins et al., J.Chromat. 202:381-390 (1980)) with 2-methoxy-4,6-dichlorotriazine thathydrolysis of the "acyl-chloride" groups to hydroxyl will occur inaqueous solutions pH 9.0 at 25° C., and thus was suggested that cyanuricchloride be used in organic solvents where no hydrolysis of thetriazinyl chlorides occur. However, organic solvents and acylatingreagents are routinely used to block the aminogroups on purines andpyrimidines, and would, therefore, make specific activation of5'-amino-tailed oligonucleotides difficult if not impossible in organicsolvents.

However, as part of the present invention it has been discovered thatamino-tailed oligonucleotides can specifically be activated in 0.1Mborate pH 8.3 with 2-methoxy-4,6-dichlorotriazine at 4° C. The activatedoligonucleotide is rapidly separated from the excess2-methoxy-4,6-dichlorotriazine at 4° C. on a Sephadex G50 straw column.The 2-methoxy-4,6-dichlorotriazinyl-oligonucleotide is then immobilizedto a polymer-coated solid phase, such as nylon beads coated with PEI, asdescribed herein.

In addition to derivatives of cyanuric chloride, other compounds maysubstitute for cyanuric chloride to specifically activate amino-tailedoligonucleotides. These include, among others, 2,4-dinitrofluorobenzene,pyrimidines (e.g., 2,4-dichloropyrimidines),2,6-dichloro-3-nitropyridine, dichloropyridiazines (e.g.,3,6-dichloropyridiazine), dichloropyrazines (e.g.,2,6-dichloropyrazine), and trichloropyrimidines. The2-amino-4,6-dichlorotriazine may also be used to activate ahydroxyl-containing solid support to yield an activated solid supportthat will have the necessary activity to react specifically with anamino-tailed oligonucleotide as described herein. Similarly,hydroxyl-containing polymers such as cellulose can be activated bycyanuric chloride and then be coated with PEI polymer. Both of the morereactive triazinyl chlorides will react with the many amino groupsavailable on the PEI polymer, and the PEI-cellulose is then reacted withactivated oligonucleotide.

Once an oligonucleotide has been activated, such as with cyanuricchloride or derivatives or substitutes mentioned above or the like, itcan be covalently linked to molecules that contain appropriatelydisposed nucleophiles such as thiols, hydroxyls, or amines. Morespecifically, in addition to covalent attachments with solid supportsdiscussed below, dichlorotriazine oligonucleotides can serve aselectrophiles for covalent attachment to many compositions including,but not limited to: proteins, enzymes, antibodies, lectins, antibodiesand protein carriers used to raise antibodies; nucleophile-derivedoligonucleotides, such as 5'- or 3'-aminohexyl-tailed oligonucleotides;nucleophile-containing polymers, such as poly(ethyleneimine),polyallylamine, and polyvinylamine; low molecular weight compoundscontaining nucleophiles, such as radioactive labels, chemiluminescentlabels, fluorescent labels, colored labels, and immunogenic labels; etc.Dichlorotriazine oligonucleotides are included in the present invention,as are processes for activating oligonucleotides with cyanuric chlorideor derivatives thereof to form the corresponding activatedoligonucleotides as discussed below.

The unreacted cyanuric chloride or derivatives thereof can be removed byexclusion chromatography or ultrafiltration, and the bead and derivedoligonucleotide conjugated wherein they are mixed together and incubatedat, preferably 20° to 50° C. for 1 to 24 hours. The residual (unreacted)amines on the bead surface can be blocked (capped) with an agent, suchas succinic anhydride, preferably in N-methyl pyrrolidone in thepresence of an appropriate base such as sodium borate, to render thesurface compatible (negatively charged) for nucleic acid hybridization.Such blocking of amines occurs through an acylating reaction or reactionof amines with an activated ester, resulting in a nonactivatable moiety.It should be noted that the ability exists for the bead surface tochemically derived such that a positive, negative, or neutral charge canbe placed on the bead.

In addition, 4,6-dichlorotriazine moieties can be replaced with thiolreactive substituents and, preferably, spacer arms are present. Thepreferred spacer arms are derived from thiol reactive substituentslinked to a 5'-tethered nucleophilic amine and are of the same formulaas listed above with the exception that Y in the formula above is athiol reactive moiety. A preferred thiol reactive moiety has a reactivegroup of either an α halo-acyl or an α, β-unsaturated carbonyl. The mostpreferred thiol reactive moieties are selected from the group consistingof haloacetamidobenzoyl and4-(N-maleimidomethyl)-cyclohexane-1-carbonyl.

A polymeric structure as described above wherein the polymeric structureis derived with thiol (sulfhydryl) containing moieties can be used inthe present invention. The actual structure of the thiol containingmoieties is non-critical as long as the thiol group or groups areavailable to react with thiol reactant moieties. The thiol chemistriesin this instance are replacements for cyanuric chloride chemistries.

The present invention also includes dipsticks having utility in nucleicacid hybridizations and comprising a nonporous solid support and a meansfor attaching a bead. Nonporous solid supports are known in the art, andthe present invention is concerned with attaching a bead to a dipstick.An example of bead attachment is the presence of a perforation orperforations (or a depression or depressions) in the dipstick whereinbeads can be attached. Preferably, perforations are employed and thebeads are attached through a pressure fit with the circumference of thehole. Such a pressure fit can occur if, for example, the circumferenceof the perforation (or depression) is slightly less than thecircumference of the bead so that the bead is pressed in place.

Preferred beads are as listed above, and may be covalently attached,either directly or through a spacer arm, to activated oligonucleotidesof the same or different sequence per a given bead, as also describedabove.

The dipstick can contain more than one bead, preferably from about twoto ten, each in their own hole, and more preferably, situated in a rowalong one edge of the dipstick. Such a dipstick can function as anindicator card, wherein multiple beads covalently attached tooligonucleotides with different sequences or specificities can beclosely aligned on a multisite dipstick, which can detect a multiplicityof pathogens in a single biological sample. A particular bead maycontain oligonucleotides with more than one nucleic acid sequence, forexample, sequences from related organisms, or a bead may only containoligonucleotides with a given nucleic acid sequence.

Numerous organisms and cell types, including pathogenic andnonpathogenic entities, can be detected in this manner from a variety ofbiological sample types. Organisms include bacteria and viruses as wellas other microorganisms, and cell types include, for example, thoseinvolved in inherited diseases and metabolic disorders. Many otherdetection applications will be apparent to one of ordinary skill in theart. For example, purported causative bacterial agents of periodontitis,such as Actinobacillus actinomycetemcomitans, Bacteroides gingivalis,Bacteroides forsythus, Bacteroides intermedius, Eikenella corrodens,Fusobacterum nucleatum, and Wolinella recta, can be identified.

It will be obvious to one of ordinary skill in the art that, althoughthe present invention is described in terms of nucleic acidhybridization assays, many other uses for these dipsticks are possible.Any member of a ligand pair can be attached to beads in the dipstick,and the dipstick can then be used to identify the corresponding ligandmember. For example, antigens or antibodies could be attached to beads,as described above, in a dipstick and then their correspondingantibodies or antigens, respectively, could be identified. In a similarmanner, biotin and streptavidin can be used.

Furthermore, the present invention also includes processes for nucleicacid detection wherein a composition comprising a polymer-coated bead,preferably having activatable amine groups, covalently attached to anactivated oligonucleotide is contacted with a target nucleic acid undersuitable conditions for hybridization and the hybridized product isdetected. Such processes can occur in a microtiter well, a flow-throughcolumn, and using a dipstick, as described above.

Target nucleic acid is usually a polynucleotide with an average lengthfrom about 20 to about 20,000 bases or nucleotides in length. Suitableconditions for hybridization refer to stringent conditions whereinbase-pairing mismatching does not occur and the hybridized product isperfectly base-paired.

The particular hybridization technique is not essential to the inventionand one of ordinary skill in the art will appreciate the variety of suchtechniques. Hybridization techniques are generally described in Hames,B. D., et al. (ed.), Nucleic Acid Hybridization, A Practical Approach,IRL Press, New York (1985). As improvements are made in hybridizationtechniques, .they can readily be applied to the present invention.

Sandwich assays can be preferably employed in the present processeswherein the target nucleic acid to be detected is either extracted or inthe original sample and is sequestered (captured) on a solid support,such as beads, by hybridization (i.e., pairing of complementary bases)to capture oligonucleotide probes covalently immobilized on the surfaceof the support. The captured nucleic acid is then hybridized to a signaloligonucleotide probe or, alternatively, this step can be performedsimultaneously with the capture of the target by including the signalprobe within, for example, the hybridization solution. The signal probecan be, for example, labeled with biotin. This results in a "sandwich"of the capture oligonucleotide probe:target nucleic acid:signaloligonucleotide probe, constituting a sandwich assay. The solid supportis then washed to remove unhybridized material, and the labeled nucleicacid is then measured in accordance with detectable characteristics ofthe label.

Various labels can be used in hybridization assays benefiting from thisinvention. Such labels act as reporter groups for detecting duplexformation between the target sequence and its complementary signalsequence. A reporter group as used herein is a group having a physicalor chemical characteristic that can be measured or detected.Detectability may be provided by such characteristics as color change,luminescence, fluorescence, or radioactivity. Or, it may be provided bythe ability of the reporter group to serve as a ligand recognition site.Any haptenic or antigenic compound can be used in combination with asuitably labeled antibody.

Enzymes of interest as reporter groups will primarily be hydrolases,particularly phosphotases, esterases, ureases, and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescers include luciferin, luminol, andoxetanediones. The above list is not complete, and the choice of labeldepends on sensitivity required, ease of conjugation with the probe,stability requirements, and available instrumentation.

In yet another embodiment, the present invention concerns methods andcompositions to increase the oligonucleotide loading capability ofcontrol pore glass (CPG) bead columns and the like in automatedoligonucleotide synthesis. The CPG bead, "burnished" glass bead, silicagel, nylon bead, etc. is activated as described above, e.g., with3-aminopropyltriethoxysilane, and the resulting aminopropyl-CPG, silicagel or glass bead is further reacted with cyanuric chloride or aderivative thereof described above. The bead or gel is then reacted withpolymer to coat the support as described above, e.g., with PEI. Theamines are then coupled with the appropriate chemistries to allowoligonucleotide synthesis. The coated bead or CPG substantiallyamplifies oligonucleotide loading on the CPG surfaces, which isparticularly important in the preparative scale synthesis ofoligonucleotides, and is a convenient substitute for expensive CPGcolumns currently used in oligonucleotide synthesis. Of course, oneskilled in the art will understand that a variety of chemistries may beused to activate the support, as described herein, as well as thesubsequent activation used to conjugate the selected polymer to thesupport, depending on the functional groups involved.

In other aspects the invention provides for the preparation of nyloncoated magnetic particles. Nylon coated magnetic particles are useful inhybridization assays, immunoassays, extractions, affinitychromatography, etc. Nylon coated magnetic beads are particularly usefulin the isolation of nucleic acids, including mRNA.

There are a number of synthetic procedures for preparing the magneticnylon particles. For example, nylon 6 (100 mg) is dissolved in m-cresol(0.7 ml) and dichloromethane (1.6 ml) (see Verschoor et al., J. Immun.Meths. 127:43-49 (1990)) or other solvents. This solution is then addedto a vigorously stirred (or sonicated) solution of γFe₂ O₃ (magneticiron oxide, obtained from Pfizer Minerals, Pigments and Metal Division,NY, N.Y.; see also U.S. Pat. No. 4,672,040, which is incorporated hereinby reference) in either 20% dimethylformamide or ethanol or water.

Alternatively, the nylon may first be dissolved and mixed with polymerdescribed above, preferably PEI polymer, and the solutionco-precipitated onto the magnetic particles. In yet another process thenylon may be activated via alkylation and reacted with the polymer. Thenylon-polymer conjugate is then dissolved and precipitated Onto themagnetic iron oxide particles, or the magnetic particles may be addedduring the polymerization reaction.

The present invention further provides means for convenient extractionand isolation of nucleic acids. Centrifuge tubes, wells or the like arecoated with a nylon-polymer coat, such as nylon-PEI as described above.This will yield a surface with multiple positive charges. The sample tobe extracted is added to the nylon-polymer coated tube and lysed andextracted using convention extraction methods, e.g., using the ISOQUICK™and the organic phase removed. When the aqueous phase is diluted, e.g.,10- to 20-fold, preferably about 15-fold, the nucleic acids in thesample adsorb to the positively charged surface of the coated tube. Theabsorbed nucleic acids can then be washed, used to perform a PCRreaction in the same tube as generally described in U.S. Pat. Nos.4,683,195 and 4,683,202, which are incorporated herein by reference, orthe nucleic acids can be eluted by a salt solution and used as desired.

The nylon-polymer coated tubes having multiple positive charges can beprepared a variety of ways. For example, as described for thenylon-polymer coated magnetic beads above, the nylon-polymer conjugate,where the polymer is preferably PEI, is dissolved and then precipitatedonto the surface of the tubes. Alternatively, nylon microbeadscovalently covered with the polymer, prepared as described above, aredissolved and precipitated onto the surface of the tubes. Similarly,polymer-coated nylon discs, filters and the like can be placed inmicrowells, centrifuge tubes, etc. for extraction and isolation ofnucleic acids.

It is to be understood that the above description and the followingexperimental section are intended to be illustrative and notrestrictive. Many variations and applications will be readily apparentto one of ordinary skill in the art upon reviewing this disclosure.

In the experimental section below, Example 1 describes the selectiveactivation of aminohexyl-tailed oligonucleotides with cyanuric chloride.Example 2 describes the comparison of nylon beads derived with variousdiamines and poly(ethyleneimine) in terms of the ability of the solidsupport to promote hybridization. Example 3 describes the use ofoligonucleotide derived nylon beads in a sandwich assay in whichhybridization is detected using an insoluble colorimetric substrate.Example 4 describes the use of oligonucleotide derived nylon beads in asandwich assay in which target is detected in complex biologicalsamples.

Example 5 describes the use of oligonucleotide derived nylon beads in asandwich in which target is detected by chemiluminescence. Example 6describes the use of oligonucleotide derived nylon beads in a sandwichin which target is detected by a fluorescence-based signal system.Example 7 describes the use of oligonucleotide derived nylon beadsimmobilized in a dipstick to form a multi-site detection panel or card.

The following Materials and Methods section pertains to theabove-summarized Examples 1-7.

MATERIALS

APB buffer is 0.18M NaCl, 0.05M Tris-HCl pH=7.6, 5 mM EDTA, and 0.5%Tween 20.

TMNZ buffer is 0.05M Tris pH=9.5, 1 mM MgCl₂, 0.5 mM ZnCl₂.

FW (filter wash) is 0.09M sodium chloride, 50 mM Tris pH 7.6, 25 mMEDTA.

SDS/FW is FW and 0.1% sodium dodecyl sulfate (SDS).

HRP (horseradish peroxidase) substrate solution is 0.1M sodium citratepH 6.5, 0.2M NaPhosphate, 0.5 mg/ml.

4-methoxy-1-naphthol, 0.02 mg/ml 3-methyl-2-benzothiazolinone hydrazoneand 0.0135% hydrogen peroxide.

AP (alkaline phosphatase) substrate solution is 1 mM5-bromo-4-chloroindoyl-3-phosphate, 1 mM nitroBlue tetrazolium, and0.01% Tween 20 in TMNZ.

Lysis and hybridization solution is 3M guanidinium thiocyanate, 2%N-lauroylsarcosine (sarcosyl), 50 mM Tris pH 7.6, 25 mM EDTA.

CAP buffer is 0.1M NaCitrate pH=6.5 and 0.2M NaPhosphate.

The fluorescent substrate for alkaline phosphatase is 0.02 mM4-methyl-umbelliferone phosphate, 0.05M Tris pH=9.5, 1 mM MgCl₂, 0.5 mMZnCl₂.

The chemiluminescent substrate for alkaline phosphatase was apre-prepared cocktail from Lumigen, Inc. (Detroit, Mich.).

Oligonucleotide sequences:

Bg1: 5'-NCAATACTCGTATCGCCCGTTATTC-3' (SEQ ID NO:1)

Aa004: 5'-NACCCATCTCTGACTTCTTCTTCGG-3' (SEQ ID NO:2)

Bg016: 5'-NTACTCGTATCGCCCGTTATTCCCG-3' (SEQ ID NO:3)

Ek007: 5'-NAAAAGTGGTATTAGCACTTCCCTT-3' (SEQ ID NO:4)

PA005: 5'-NGACATACCTTCCACCATCTGCAAG-3' (SEQ ID NO:5)

PA505: 5'-NCTTGCAGATGGTGGAAGGTATCTC-3' (SEQ ID NO:6)

UP9A: 5'-NCTGCTGCCTCCCGTAGGAGT-3' (SEQ ID NO:7)

UP007: 5'-NGTATTACCGCGGCTGCTG-3' (SEQ ID NO:8)

Poly(ethyleneimine) was purchased from Polysciences (Warrington, Pa.).

Burnished or unpolished nylon beads were purchased from Precision BallCompany (Chicago, Ill.) and The Hoover Group (Sault St. Marie, Mich.)

Triethyloxonium tetrafluoroborate, hexanediamine, phenylenediamine,succinic anhydride and N-methyl-pyrrolidinone (N-methyl-pyrrolidone,m-pyrol) were purchased from Aldrich Chemical (Milwaukee, Ill.).

N-succinimidyl 4-(iodoacetamido)-benzoate (SIAB) and Tween 20 waspurchased from Pierce (Rockford, Ill.).

Guanidium isothioscyanate (GuSCN) was purchased form Kodak (Rochester,N.Y.).

Nylon membrane, NYTRAN™, was purchased from Scheicher & Schuell, (Keene,N.H.).

The di- and triamines EDR-148, ED-400, ED-6000, and T-3000 were a giftfrom Texaco Chemical Company, (Houston, Tex.).

PROCEDURES Oligonucleotide Synthesis

Oligonucleotides complementary to regions conserved or hypervariableregions of the 16S-ribosomal RNA of either Actinobacillusactinomycetemcomitans (Aa), Bacteroides gingivalis (Bg), Bacteroidesintermedius (Bi), Eikenella corrodens (Ek), Fusobacterium nucleatum(Fn), or Wolinella recta (Wr) were synthesized using phosphoramiditechemistry on either an ABI 380B or a Milligen 7500 automated DNAsynthesizer. The oligonucleotides were prepared using the standardphosphoramidite chemistry supplied by the vendor or the H-phosphonatechemistry. Appropriately blocked dA, dG, dC, and T phosphoramidites arecommercially available in these forms, and synthetic nucleosides mayreadily be converted to the appropriate form. Oligonucleotides werepurified by adaptations of standard methods. Oligonucleotides with5'-trityl groups were chromatographed on HPLC using a 12 μm, 300 ÅRainin (Woburn, Mass.) Dynamax C-8 4.2×250 mm reverse phase column usinga gradient of 15% to 55% MeCN in 0.1N Et₃ NH⁺ OAc⁻, pH 7.0, over 20 min.When detritylation was performed, the oligonucleotides were furtherpurified by gel exclusion chromatography. Analytical checks for thequality of the oligonucleotides were conducted with a Toso-Haas DEAE-NPRcolumn at alkaline pH and by polyacrylamide gel electrophoresis (PAGE).

Preparation of the Polymer-coated Nylon Bead

25,000 3/32 inch diameter unpolished nylon beads were placed in a flaskcontaining 1800 ml of 100% anhydrous n-methyl-pyrrolidinone and mixedfor 5 minutes at ambient temperature. 200 ml of 1 molar triethyloxoniumtetrafluoroborate in dichloromethane was added and the mixture wasstirred for 30 minutes at ambient temperature. The beads were thendecanted and washed quickly with 4, 500 ml changes of 100%n-methyl-pyrrolidinone. The beads were then transferred to a solutionconsisting of 3% w/v 10,000MW poly(ethyleneimine), prepared from a 30%aqueous solution of poly(ethyleneimine), in n-methyl-pyrrolidone andstirred for 12 to 24 hours at ambient temperature. The beads were washedwith 2000 ml n-methyl-pyrrolidone, 1000 ml SDS/FW and finally 10×2 literstilled water. The beads were then dried under a high vacuum for 4 to 5hours without the use of heat. The amine content of the beads wasdetermined by reaction with picyrlsulfonic acid.

Preparation of Cyanuric Chloride-derived Oligonucleotides

10 to 1000 μg of 5'-amine-linked oligonucleotide were reacted with anexcess of recrystallized cyanuric chloride in 10% n-methyl-pyrrolidonein an alkaline buffer (pH 8.3 to 8.5, preferably) at 19° to 25° C. for30 to 120 minutes. The final reaction conditions consisted of 0.15Msodium borate at pH 8.3, 2 mg/ml recrystallized cyanuric chloride and500 ug/ml respective aminohexyl oligonucleotide. The unreacted cyanuricchloride was removed by size exclusion chromatography on a G-50Sephadex™ (Pharmacia, Uppsala, Sweden) column.

Preparation of Iodoacetamidobenzoylated Oligonucleotides

100 to 1000 μg of 5'-amine-linked oligonucleotide (UP9A) oligonucleotidewere reacted with an excess of N-succinimidyl 4-(iodoacetamido)-benzoate(SIAB) in an alkaline (pH 8.0 preferably) buffer at 18° to 25° C. for 30to 120 minutes. The unreacted SIAB is removed by size exclusionchromatography on G-50 SEPHADEX™ (Pharmacia, Uppsala, Sweden).

Preparation of Oligonucleotide Derived Nylon Beads

For cyanuric chloride derived oligonucleotides, poly(ethyleneimine)coated nylon beads described above were placed in a volume of 0.1Msodium borate pH 8.3 equal to the volume of the beads at 4° C. Thepurified cyanuric chloride derived oligonucleotide was then added to thebeads, and the mixture was vigorously agitated at ambient temperature(19° to 23° C.) for 60 minutes. The beads were then washed twice with0.1M sodium borate pH 8.3. Succinic anhydride was then added at aconcentration of 10 mg/ml in 90% N-methyl-pyrrolidone, 10% 1M sodiumborate pH 8.3 with a volume three times that the volume of the beads.The reaction was allowed to proceed for 1 hour at ambient temperature.The beads were then washed 3 times with 250 ml of 100%N-methyl-pyrrolidone, twice with distilled water, 5 times with 250 mlSDS/FW and then 4 times with 1 liter of distilled water. Beads werestored dry or in 25 mM EDTA. Radioactivity per bead was determined byliquid scintillation counting.

For iodoacetamidobenzoylated oligonucleotides, poly(ethyleneimine)coated nylon beads described above were placed in a volume of 0.1Msodium borate pH 8.3 equal to the volume of the beads, and iminothiolanewas added to a final volume of 5 mg/ml. The beads were allowed to reactfor 1 hour at ambient temperature and then washed 10 times with 0.1Msodium borate pH 8.3 and 10 mM EDTA at 4° C. The thiolatedpoly(ethyleneimine) coated nylon beads described above were placed in avolume of 0.1M sodium borate pH 8.3 and 25 mM EDTA equal to the volumeof the beads at 4° C. The purified iodoacetamidobenzoylatedoligonucleotides were then added to the beads, and the mixture wasvigorously agitated at ambient temperature (19° to 23° C.) for 4 hours.The beads were then washed twice with 0.1M sodium borate and 25 mM EDTAand then incubated with a volume three times that of the beads with 10mg/ml iodoacetamide in a 1:1 v/v ratio N-methyl-pyrrolidone and 0.1Msodium borate pH 8.3. The reaction was allowed to proceed at ambienttemperature for 1 hour. The beads were then washed 3 times with 250 mlof 100% N-methyl-pyrrolidone, twice with distilled water, 5 times with250 ml SDS/FW and then 4 times with 1 liter of distilled water. Beadswere stored dry or in 25 mM EDTA. Radioactivity per bead was thendetermined by liquid scintillation counting.

Preparation of the Membrane Solid Support

A 16 cm² piece of NYTRAN™ (Scheicher & Schuell, Keene, N.H.) wasincubated with 10 ml of 5 mg/ml iminothiolane in 0.1M sodium borate atpH=8.3 for 30 minutes at ambient temperature. The membrane was washedwith 5 changes of the sodium borate buffer described above. Theintroduced thiol groups were determined using5,5-dithio-bis(2-nitrobenzoic acid). The derived membrane was then cutinto 0.28 cm² discs and washed once with 0.1M sodium borate buffer.IAB-oligonucleotide was prepared as described above and mixed with themembrane discs. 300 membrane discs were submerged in 2 ml of 0.1M sodiumborate buffer containing 1.0 mg of IAB-oligonucleotide, and the reactionwas allowed to proceed at room temperature with constant agitation for16 hours in the dark. The discs were then washed sequentially with 0.1Msodium borate, SDS/FW. 1.2 micrograms of Bg5B oligonucleotide was boundper filter disc. The unreacted thiol groups were blocked with 50 mg/mliodoacetamide in 0.1M sodium borate pH=8.3. The filters were then washedfurther with sodium borate and SDS/FW.

Lysis of Bacteria and Hybridization Conditions

1×10⁸ Cells of Bacteroides gingivalis (Bg) were lysed in 100 μL of lysissolution at 19° C. The cell lysate was then heated in an 65 degree waterbath for 10 minutes. Biotinylated probe was added to the lysate solutionand to the diluent (GuSCN lysis solution) to a final concentration of100 ng/mL, and 5 to 8 5-fold serial dilutions were made of the startinglysate. The solutions were incubated with either the derived nylon beador the Nytran™ (Scheicher & Schuell, Keene, N.H.) that had beencovalently immobilized with 0.1 μg of respective oligonucleotide probe(capture probe) for 1 hour at ambient temperature with mild agitation.The solid supports were then washed once with the lysis andhybridization solution, once with FW, and once with SDS/FW.Streptavidin/HRP conjugate was added to a final concentration of 1microgram/ml (based on streptavidin) in SDS/FW and incubated 10 to 15minutes at ambient temperature with mild agitation. The beads andfilters were then washed three times with SDS/FW and then once with CAPbuffer. 4-methoxy-naphthol napthol substrate solution described abovewas added, and the reaction was allowed to proceed for 15 minutes atambient temperature. The beads or filters were then quickly washed oncewith SDS/FW and then once with FW and allowed to air dry in the dark.Quantitative determination of the extent of hybridization (capture oftarget nucleic acid) using insoluble substrates for either horseradishperoxidase or alkaline phosphatase:

After the completion of the sandwich assay on the solid support, herein3/32 inch nylon beads, and the deposition of the insoluble substrateproduct onto the surface of the bead described above for either HRP oralkaline phosphatase, the quantity of target captured was determined byfluorescence quenching. The beads were dried for 15 to 30 minutes atambient temperature and then individually placed in a round bottomopaque white microtiter plate (Dynatek Laboratories, Chantilly, Va.).The beads were then read using a fluorometer (Fluoroskan II, FlowLaboratories, McLean, Va.) in which excitation was at 350 nanometers andemmision was at 456 namometers. The beads possessed an intrinsicfluorescence of about 800 relative fluorescence units, and the presenceof the colorimetric substrate product effectively quenched theinstrinsic fluorescence. The lower the indicated fluorescence correlatedwith the greater the quantity of captured target nucleic acid.

EXAMPLE 1

Example 1 describes the selective modification and activation of thetethered 5'-amine of oligonucleotides with cyanuric chloride. It isshown that the derivitization of the oligonucleotide occurs only at thetethered amine.

The sequence UP9A, which either possessed a 5'-aminohexyl tail or didnot possess a 5'-aminohexyl tail, was compared with respect toreactivity with cyanuric chloride. 50 ug of each type oligonucleotidewas reacted in a 400 ul volume containing 0.15M sodium borate pH=8.3, 2mg/ml cyanuric chloride (from 50 mg/ml freshly prepared stock in 100%acetonitrile). The reaction was allowed to occur at 19° C. for 30minutes. The reaction mixture was then analyzed by C-18 reverse phaseHPLC utilizing a 5 to 45% acetonitrile gradient in TEA. Thechromatographs from the respective reaction mixtures and both types ofstarting oligonucleotide are shown in FIGS. 3A, 3B, 3C and 3D.

The non-tailed oligonucleotide sequence UP9A is shown in panel A andelutes off the column at 9.025 minutes whereas the amine-tailedoligonucleotide sequence UP9A elutes at 9.205 minutes (panel B). Thechromatograph in panel C shows that the non-tailed oligonucleotidesequence UP9A does not react with cyanuric chloride, as theoligonucleotide continues to be eluted at 9.005 minutes. In panel D, theamine-tailed oligonucleotide sequence UP9A reacts almost to completionwith cyanuric chloride resulting in a dichlorotriazine derivitive thatis eluted at 11.6 minutes, almost 2.5 minutes later than the UP9Aamine-tailed starting material. The profiles indicate that only the UP9Asequence possessing the 5'-tethered amine reacted with cyanuric chloridedemonstrating that cyanuric chloride reacted selectively with the amineand not with any of the sugars or bases present in the oligonucleotide.Therefore, it was shown that 5'-aminohexyl oligonucleotides areselectively activated with cyanuric chloride, resulting in a probe isimmobilized only at the 5'-end onto a solid support.

EXAMPLE 2

This example describes the derivation of nylon beads with several typesof diamines and poly(ethyleneimine), and then the subsequent attachmentof 4,6-dichlorotriazine oligonucleotides. A comparison of thehybridization properties of the respective beads is also described.

200 bead batches were derived with either hexanediamine, Jeffamine EDR148, 1,4-phenylenediamine, Jeffamine T3000, or poly(ethyleneimine) usingthe procedure described above relating to preparation of polymer-coatedbeads. Each diamine or triamine bead type contained between 200 nmolesto 2 umoles of amine and the poly(ethyleneimine) contained approximately100 nmoles of amine.

Each bead type was then reacted with 4,6-dichlorotriazine activatedoligonucleotide (Bg5 sequence) as described above. The unreacted amineswere then blocked with succinnic anhydride as described above and testedin a hybridization assay as described above. The phenylenediamine beadsshowed no ability to capture target nucleic acid whereas the JeffaminesEDR-148, T-3000, and hexanediamine type beads captured nucleic acidtarget but at a rate about 25-fold less than that of thepoly(ethyleneimine) beads as determined by the fluorescence quenchingassay described above. The results are summarized in the table below.

    ______________________________________                                        Bead type:        Lower limit of detection:                                   ______________________________________                                        Phenylenediamine  no target detected                                          EDR-148           5 × 10.sup.7 cells                                    T-3000            2.5 × 10.sup.8 cells                                  Hexanediamine     2.5 × 10.sup.8 cells                                  Poly(ethyleneimine)                                                                             2 × 10.sup.6 cells                                    ______________________________________                                    

The results indicate that polymer-coated beads were approximately25-fold more efficient in the capture of target nucleic acid andtherefore present the best surface type for covalent immobilization ofoligonucleotides and capture of target nucleic acid.

EXAMPLE 3

Example 3 compares the solid supports formed from Nytran™ (Scheicher &Schuell, Keene, N.H.) membranes and nylon beads in a sandwich assayformat in which a target nucleic acid sequence is sequestered and thendetected using a colorimetric assay format.

3M GnSCN lysis solution was used to lyse 1×10⁸ cells of Actinobacillusactinomycetemcomitans (Aa), Bacteroides gingivalis (Bg), Bacteroidesintermedius (Bi), Eikenella corrodens (Ec), Fusobacterium nucleatum(Fn), and Wolinella recta (Wr) in 100 microliter volumes at 19° C. Thelysate was then heated to 65° C. for 5 minutes. A biotinylated 24-meroligonucleotide probe complementary to conserved regions of bacterial16s rRNA (signal probe) was added to a final concentration of 100nanograms per ml.

5-fold serial dilutions of the lysates were made using diluentscontaining the biotinylated signal oligonucleotides and 1×10⁸ totalcells of Aa, Bi, Ek, Fn, and Wr. The solutions were then incubated for30 minutes at ambient temperature with NYTRAN™ (Scheicher & Schuell,Keene, N.H.) discs or 2 nylon beads that had covalently immobilized 0.1μg of Bg1 specific oligonucleotide probe (capture probe). The solidsupports were washed with SDS/FW at ambient temperature and thenincubated with 10 ng/ml of streptavidin/horseradish peroxidase (SA/HRP)conjugate in SDS/FW for 5 minutes at ambient temperature. The solidsupports were then washed with SDS/FW, FW, and then the presence ofperoxidase was determined by incubating the filter with the HRPsubstrate solution described above to form an insoluble product.

The results indicated that, in the 30 minute hybridization, 2×10⁵ cellswere detected using the nylon beads as solid supports whereas 1×10⁶cells were detected using NYTRAN™ (Scheicher & Schuell, Keene, N.H.)solid supports as determined by the fluorescence quenching assaydescribed above. The control in which Aa, Bi, Ek, and Wr cells werepresent and Bg was absent showed no color, indicating that the captureof Bg was specific. It was also noted that most HRP insoluble substratesfaded upon dying on the NYTRAN™ (Scheicher & Schuell, Keene, N.H.)membranes, whereas no fading occurred on the nylon beads.

EXAMPLE 4

Example 4 compares the solid supports formed from NYTRAN™ (Scheicher &Schuell, Keene, N.H.) membranes and nylon beads in a sandwich assayformat in which a target nucleic acid sequence is sequestered and thendetected in a complex biological sample containing whole blood.

3M GnSCN lysis solution was used to lyse 1×10⁸ cells of Bacteroidesgingivalis (Bg) spiked into a plaque sample containing visiblequantities of blood (approximately 25 microliters packed cell volume) in250 microliter volume at 19° C. and then split into two equal volumes.The lysate was then heated to 65° C. for 5 minutes. A biotinylated24-mer oligonucleotide probe complementary to conserved regions ofbacterial 16s rRNA (signal probe) was added to a final concentration of100 nanograms per ml.

5-fold serial dilutions of the lysates were made using diluents in 3MGuSCN lysing and hybridization solution containing the biotinylatedsignal oligonucleotides and one part in ten of whole blood. Thesolutions were then incubated for 30 minutes at ambient temperature witha Nytran™ (Scheicher & Schuell, Keene, N.H.) discs or 2 nylon beads thathad covalently immobilized 0.1 μg of Bg1 specific oligonucleotide probe(capture probe). The solid supports were then washed with SDS/FW atambient temperature and incubated with 10 ng/ml ofstreptavidin/horseradish peroxidase (SA/HRP) conjugate in SDS/FW for 5minutes at ambient temperature. The solid supports were then washed withSDS/FW, FW, and then the presence of peroxidase was determined byincubating the filter with the HRP substrate solution described above toform an insoluble product.

The results indicated that, in the 30 minute hybridization, 8×10⁵ cellswere detected using the nylon beads as solid supports whereas 4×10⁶cells were detected using NYTRAN™ (Scheicher & Schuell, Keene, N.H.)solid supports as determined by the fluorescence quenching assaydescribed above. More importantly, the NYTRAN™ (Scheicher & Schuell,Keene, N.H.) filters were significantly stained with lysed bloodproducts whereas the nylon beads retained their starting color.

EXAMPLE 5

Example 5 compares the solid supports formed from NYTRAN™ (Scheicher &Schuell, Keene, N.H.) membranes and nylon beads in a sandwich assayformat in which a target nucleic acid sequence is sequestered and thendetected using a chemiluminescence assay format.

3M GnSCN lysis solution was used to lyse 1×10⁸ cells of Actinobacillusactinomycetemcomitans (Aa), Bacteroides gingivalis (Bg), Bacteroidesintermedius (Bi), Eikenella corrodens (Ec), Fusobacterium nucleatum(Fn), and Wolinella recta (Wr) in 100 microliter volumes at 19° C. Thelysate was then heated to 65° C. for 5 minutes. A biotinylated 24-meroligonucleotide probe complementary to conserved regions of bacterial16s rRNA (signal probe) was added to a final concentration of 100nanograms per ml.

5-fold serial dilutions of the lysates were made using diluents in 3MGuSCN lysing and hybridization solution containing the biotinylatedsignal oligonucleotides and 1×10⁸ total cells of Aa, Bi, Ek, Fn, and Wr.The solutions were then incubated for 30 minutes at ambient temperaturewith NYTRAN™ (Scheicher & Schuell, Keene, N.H.) discs or 2 nylon beadsthat had covalently immobilized 0.1 μg of Bg1 specific oligonucleotideprobe (capture probe). The solid supports were washed with SDS/FW atambient temperature following by washing with 0.5% Tween 20, 1 mM MgCl₂,0.01M Tris-HCl pH 8.0 (APB) and then incubated with 0.4 ug/ml ofstreptavidin/alkaline phosphatase (SA/AP) conjugate in APB for 5 minutesat ambient temperature. The solid supports were washed 5 times with APB,TMNZ, and then the presence of alkaline phosphatase was determined byincubating either the NYTRAN™ (Scheicher & Schuell, Keene, N.H.) filtersor the nylon beads with 200 microliters of Lumigen (from Lumigen, Inc.,Detroit, Mich.) in 5 mm×40 mm polypropylene tubes. The results are shownin the table below.

    ______________________________________                                                   Chemiluminescent Signal                                            Cell number:                                                                             NYTRAN ™ solid supports:                                                                    Nylon beads:                                      ______________________________________                                        1 × 10.sup.8                                                                       off scale        off scale                                         2 × 10.sup.7                                                                       1600             off scale                                         4 × 10.sup.6                                                                       1750             1650                                              8 × 10.sup.5                                                                       1700             680                                               1.6 × 10.sup.5                                                                     1600             320                                               3.2 × 10.sup.4                                                                     1800             260                                               6.4 × 10.sup.3                                                                     1800             210                                               control    1700             200                                               ______________________________________                                    

Therefore, the results indicate that, in the 30 minute hybridization,3×10⁴ cells were detected using the nylon beads as solid supportswhereas only 1×10⁸ cells were detected using the NYTRAN™ (Scheicher &Schuell, Keene, N.H.) solid supports. This approximately 10,000-folddifference in the lower level of detection of the target was due to thesevere background of nonspecific binding of the alkaline phosphatase tothe NYTRAN™ (Scheicher & Schuell, Keene, N.H.) filters. The nylon beads,therefore, allowed the sensitive detection of Bg 16s rRNA using achemiluminescence based signal system.

EXAMPLE 6

Example 6 demonstrates the nylon solid supports in a sandwich assayformat in which a target nucleic acid sequence is sequestered and thendetected using a fluorescence-based assay format.

3M GnSCN lysis solution was used to lyse 1×10⁸ cells of Actinobacillusactinomycetemcomitans (Aa), Bacteroides gingivalis (Bg), Bacteroidesintermedius (Bi), Eikenella corrodens (Ec), Fusobacterium nucleatum(Fn), and Wolinella recta (Wr) in 100 microliter volumes at 19° C. Thelysate was heated to 65° C. for 5 minutes. A biotinylated 24-meroligonucleotide probe complementary to conserved regions of bacterial16s rRNA (signal probe) was added to a final concentration of 100nanograms per ml.

5-fold serial dilutions of the lysates were made using diluents in 3MGuSCN lysing and hybridization solution containing the biotinylatedsignal oligonucleotides and 1×10⁸ total cells of Aa, Bi, Ek, Fn, and Wr.The solutions were then incubated for 30 minutes at ambient temperaturewith 2 black nylon beads prepared by The Hoover Group (Sault St. Marie,Mich.) that had covalently immobilized 0.1 μg of Bg1 specificoligonucleotide probe (capture probe). The solid supports were washedwith SDS/FW at ambient temperature following by washing with 0.5%TWEEN-20™, 1 mM MgCl₂, 0.01M Tris-HCl pH 8.0 (APB) and then incubatedwith 0.4 ug/ml of streptavidin/alkaline phosphatase (SA/AP) conjugate inAPB for 5 minutes at ambient temperature. The solid supports were thenwashed 5 times with APB, TMNZ, and then the presence of alkalinephosphatase was determined by incubating the nylon beads with 150microliters of 0.5 mM 4-methyl-umbelliferyl phosphate (4-hydroxymethylcoumarin) in black microtiter well strips (Dynatek, Laboratories,Chantilly, Va.). Incubation was for 30 minutes at 37° C. The plates werethen directly read using a Fluoroskan II fluorometer (Flow Laboratories,McLean, Va.) using an excitation wavelength of 360 nm and an emissionwavelength of 456 nm. The results are shown in the table below.

    ______________________________________                                                     Fluorescent Signal                                               Cell number: Nylon beads                                                      ______________________________________                                        1 × 10.sup.8                                                                         1250                                                             2 × 10.sup.7                                                                         1980                                                             4 × 10.sup.6                                                                         680                                                              8 × 10.sup.5                                                                         175                                                              1.6 × 10.sup.5                                                                       58                                                               3.2 × 10.sup.4                                                                       26                                                               6.4 × 10.sup.3                                                                       23                                                               control      18                                                               ______________________________________                                    

The results indicate that, in the 30 minute hybridization, 6×10³ cellswere detected using the nylon beads as solid supports. The nylon beads,therefore, allowed the sensitive detection of Bg 16s rRNA using afluorescence based signal system.

EXAMPLE 7

Example 7 describes the use of 3/32 inch nylon beads immobilized in anonporous, plastic card to form a multi-panel detection compositiondescribed herein as a dipstick. The dipstick (or indicator card) soformed possesses multiple and distinct sites that allow the specificdetection of multiple pathogens in a single sample. In this Example, thespecific detection of Bg and Ek is demonstrated.

A set of six identical dipsticks were prepared, in which the generalconfiguration of the dipstick is shown in FIGS. 1A, 1B and 2. The fivebeads, each possessing a different capture oligonucleotide, were placedin the dipstick from left to right in the following order:

1: PA005 (positive control)

2: Aa004 (for the detection of Aa rRNA)

3: Bg002 (for the detection of Bg rRNA)

4: Ek007 (for the detection of Ek rRNA)

5: PA505 (negative control)

Each dipstick was tested in 400 ul lysis and hybridization solutiondescribed above containing biotinylated UP9A and UP007 at 500 ng/ml,PA505 at 5 ng/ml, and either:

1) 1×10⁸ Bg cells.

2) 2×10⁷ Bg cells.

3) 1×10⁸ Ek cells.

4) 2×10⁷ Ek cells.

5) 1×10⁸ Bg cells and 1×10⁸ Ek cells.

6) 2×10⁸ Bg cells and 2×10⁸ Ek cells.

The dipsticks were processed sequentially through the followingsolutions with constant agitation at 2 hertz: 10 minutes in thehybridization solution, 2 minutes in SDS/FW, 5 minutes in SA/HRPconjugate, 2 minutes in SDS/FW, 2 minutes in CAP buffer, and thendeveloped 10 minutes in 4MN substrate solution. The results aredescribed in the following table.

    ______________________________________                                                  Solution:                                                           Bead number:                                                                              1     2        3   4      5   6                                   ______________________________________                                        1           ++    ++       ++  ++     ++  ++                                  2           -     -        -   -      -   -                                   3           ++    +        -   -      ++  +                                   4           -     -        ++  +      ++  +                                   5           -     -        -   -      -   -                                   ______________________________________                                         wherein ++ indicates strong colorimetric signal, + indicates medium           colorimetric signal, - indicates no signal. The results indicate that the     dipstick was able to detect specifically the presence of Bg and Ek.      

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "N = nucleotide with                            5'- terminal phosphate O modified with -(CH-2)-n - X - Y,                     NH- or -NHC:O(CH-2)-mNH-,                                                     m = 2-12 inclusive; Y = 4,6-dichlorotriazine or                               thiol reactive moiety"                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       NCAATACTCGTATCGCCCGTTATTC25                                                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "N = nucleotide with                            5'- terminal phosphate O modified with -(CH-2)-n - X - Y,                     NH- or -NHC:O(CH-2)-mNH-,                                                     m = 2-12 inclusive; Y = 4,6-dichlorotriazine or thiol                         reactive moiety"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       NACCCATCTCTGACTTCTTCTTCGG25                                                   (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "N = nucleotide with                            5'- terminal phosphate O modified with -(CH-2)-n - X - Y,                     NH- or -NHC:O(CH-2)-mNH-,                                                     m = 2-12 inclusive; Y = 4,6-dichlorotriazine or thiol                         reactive moiety"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       NTACTCGTATCGCCCGTTATTCCCG25                                                   (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "N = nucleotide with                            5'- terminal phosphate modified with -(CH-2)-n - X - Y,                       NH- or -NHC:O(CH-2)-mNH-,                                                     m = 2-12 inclusive; Y = 4,6-dichlorotriazine or thiol                         reactive moiety"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       NAAAAGTGGTATTAGCACTTCCCTT25                                                   (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "N = nucleotide with                            5'- terminal phosphate O modified with -(CH-2)-n - X - Y,                     NH- or -NHC:O(CH-2)-mNH-,                                                     m = 2-12 inclusive; Y = 4,6-dichlorotriazine or thiol                         reactive moiety"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       NGACATACCTTCCACCATCTGCAAG25                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "N = nucleotide with                            5'- terminal phosphate O modified with -(CH-2)-n - X - Y,                     NH- or -NHC:O(CH-2)-mNH-,                                                     m = 2-12 inclusive; Y = 4,6-dichlorotriazine or thiol                         reactive moiety"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       NCTTGCAGATGGTGGAAGGTATCTC25                                                   (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "N = nucleotide with                            5'- terminal phosphate O modified with -(CH-2)-n - X - Y,                     NH- or -NHC:O(CH-2)-mNH-, m = 2-12                                            inclusive; Y = 4,6-dichlorotriazine or thiol reactive moi                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       NCTGCTGCCTCCCGTAGGAGT21                                                       (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "N = nucleotide with                            5'- terminal phosphate O modified with -(CH-2)-n - X - Y,                     NH- or -NHC:O(CH-2)-mNH-,                                                     m = 2-12 inclusive; Y = 4,6-dichlorotriazine or thiol                         reactive moiety"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       NGTATTACCGCGGCTGCTG19                                                         __________________________________________________________________________

What is claimed is:
 1. A composition comprising an oligonucleotidecovalently attached to amine-containing polymer-coated solid supportwherein the covalent attachment occurs between the amine of thepolymer-coated solid support and an amine which is tethered to theoligonucleotide.
 2. The composition of claim 1, wherein the solidsupport is comprised of nylon, polystyrene, glass, latex, polypropyleneor activated cellulose.
 3. The composition of claim 1, wherein the solidsupport is comprised of nylon.
 4. The composition of claim 1, whereinthe solid support is a bead, membrane, microwell, centrifuge tube, orstring.
 5. The composition according to claim 1 wherein the polymer ispoly(ethyleneimine), polyvinylamine, polyallylamine,poly(acryhydrazide), poly(p-aminostyrene), or poly-L-lysine.
 6. Thecomposition according to claim 1 wherein unreacted amines of the polymerare blocked.
 7. The composition according to claim 6, wherein theunreacted amines are blocked with succinic anhydride.
 8. The compositionof claim 1, wherein the oligonucleotide is activated with cyanuricchloride or a derivative thereof.
 9. The composition according to claim1, wherein the oligonucleotide is linked to the polymer coated solidsupport through an amine which is tethered to the 5' or 3' end of theoligonucleotide.
 10. The composition according to claim 1 wherein theoligonucleotide is linked to the polymer coated solid support through anamine which is tethered to the oligonucleotide at a location between the5' and 3' ends of the oligonucleotide.
 11. A method for producing anoligonucleotide covalently linked to a amine-containing polymer, throughan amine which is tethered to the 5' end or the 3' end or at a locationbetween the 5' end and 3' end of the oligonucleotide, which methodcomprises:activating an alkylamine-containing oligonucleotide withcyanuric chloride or a derivative thereof, and conjugating the activatedoligonucleotide to an amine-containing polymer.
 12. The method accordingto claim 11, wherein the cyanuric chloride derivative is2-methoxy-4,6-dichlorotriazine, 2,4,6-tribromotriazine,2,4,6-triiodoatrazine, 2-monochloro-4,6-diiodotriazine, or2-amino-4,6-dichlorotriazine.
 13. The method according to claim 11,wherein the polymer is poly(ethyleneimine), polyvinylamine,polyallylamine, poly(acryhydrazide), poly(p-aminostyrene), orpoly-L-lysine.
 14. The method according to claim 13, wherein the polymeris covalently linked to a solid support.
 15. The method according toclaim 14, wherein the solid support is a bead, membrane, microwell,centrifuge tube, or string.
 16. The method according to claim 11,further comprising the step of blocking the unreacted amines of thepolymer.
 17. A method for preparing a support for use in oligonucleotidesynthesis, comprisingactivating a control pore glass, silica gel ornylon bead support with 3-aminopropyltriethoxysilane; reacting theactivated support with cyanuric chloride or derivatives thereof; andconjugating the cyanuric chloride reacted support to an amine-containingpolymer of poly(ethyleneimine), polyvinylamine, polyallylamine, orpoly(p-aminostyrene).
 18. The composition of claim 8 wherein thecyanuric chloride derivative is selected from the group consisting of2-methoxy-4,6-dichlorotriazine, 2,4,6-tribromotriazine,2,4,6-triiodotriazine, 2-monochloro-4,6-diiodotriazine, and2-amino-4,6-dichlorotriazine.
 19. The composition according to claim 1,wherein the oligonucleotide is activated with 4,2-dinitrofluorobenzene,2,4, dichloropyrimidine, 2,6-dichloro-3-nitropyridine,3,6-dichloropyridiazine, or 2,6-dichloropyrazine.